Subtractive cDNA Library Research Articles

Redox homeostasis at SAM: a new role of HINT protein.

ScHINT1 was identified at sugarcane SAM using subtractive libraries. Here, by bioinformatic tools, two-hybrid approach, and biochemical assays, we proposed that its role might be associated to control redox homeostasis. Such control is important for plant development and flowering transition, and this is ensured with some protein partners such as PAL and SBT that interact with ScHINT1. The shoot apical meristem transition from vegetative to reproductive is a crucial step for plants. In sugarcane (Saccharum spp.), this process is not well known, and it has an important impact on production due to field reduction. In view of this, ScHINT1 (Sugarcane HISTIDINE TRIAD NUCLEOTIDE-BINDING PROTEIN) was identified previously by subtractive cDNA libraries using Shoot Apical Meristem (SAM) by our group. This protein is a member of the HIT superfamily that was composed of hydrolase with an AMP site ligation. To better understand the role of ScHINT1 in sugarcane flowering, here its function in SAM was characterized using different approaches such as bioinformatics, two-hybrid assays, transgenic plants, and biochemical assays. ScHINT1 was conserved in plants, and it was grouped into four clades (HINT1, HINT2, HINT3, and HINT4). The 3D model proposed that ScHINT1 might be active as it was able to ligate to AMP subtract. Moreover, the two-hybrid approach identified two protein interactions: subtilase and phenylalanine ammonia-lyase. The evolutionary tree highlighted the relationships that each sequence has with specific subfamilies and different proteins. The 3D models constructed reveal structure conservation when compared with other PDB-related crystals, which indicates probable functional activity for the sugarcane models assessed. The interactome analysis showed a connection to different proteins that have antioxidative functions in apical meristems. Lastly, the transgenic plants with 35S::ScHINT1_AS (anti-sense orientation) produced more flowers than wild-type or 35S::ScHINT1_S (sense). Alpha-tocopherol and antioxidant enzymes measurement showed that their levels were higher in 35S::ScHINT_S plants than in 35S::ScHINT1_AS or wild-type plants. These results proposed that ScHINT1 might have an important role with other proteins in orchestrating this complex network for plant development and flowering.

Analysis of Differentially Expressed Rice Genes Reveals the ATP-Binding Cassette Transporters as Candidate Genes Against the Sheath Blight Pathogen, Rhizoctonia solani

Sheath blight is a serious rice disease worldwide, and genes involved in resistance remain unclear. In the present study, a virulent field isolate of Rhizoctonia solani was used to inoculate detached leaves of a sheath blight-resistant rice cultivar, Jasmine 85; a suppression subtractive cDNA library was constructed using RNA isolated 16 h postinoculation (hpi); and differentially expressed genes were identified from the cDNA library. A total of 159 uniquely expressed sequence tags were identified, including 105 from rice with enrichment in categories related to cellular response, molecular signaling, and host defense. Coupled with gene expression studies by DNA microarray, 27 highly induced genes involved in signal transduction and defense responses were identified within 16 hpi. Three members of the ATP-binding cassette (ABC) transporter gene family ( OsABC1, OsABC9, and OsABC12) encoding pleiotropic drug resistance-like ABC transporters were mapped to different sheath blight resistance quantitative trait loci (QTLs), and their differential expressions were validated. Three high-resolution melting (HRM) markers were developed from these ABC gene family members to distinguish alleles between sheath blight-susceptible cultivar Lemont and resistant cultivar Jasmine 85. Association of sheath blight resistance to these HRM markers was examined in 77 recombinant inbred lines derived from the cross between Jasmine 85 and Lemont. The OsABC9 gene located in a major sheath blight resistance QTL qShB9-2 showed a major contribution to sheath blight resistance. These results are useful for marker-assisted selection and functional validation of the ABC genes in sheath blight disease resistance. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.

Differentially Expressed Long-Term Salinity Responsive Sequences in Halophyte Suaeda maritima (L.) Dumort

Salinity is a major abiotic stress that affects plant growth and productivity. To cope with salt stress, plants express large number of salt responsive genes and proteins that are involved in a wide range of cellular functions. In the present study, halophytic plant Suaeda maritima (L.) Dumort. were hydroponically exposed to NaCl for understanding the molecular mechanisms behind salinity tolerance using PCR-based Suppressive Subtractive Hybridization (SSH). Two cDNA subtraction libraries were constructed between Suaeda maritima X Sesuvium portulacastrum and Suaeda maritima X Salicornia brachiata to identify differentially expressed genes from leaves exposed to 200mM NaCl treatment for 14 days. A total of 224 clones from both libraries were assembled into 109 unique-ESTs grouped into different functional categories. Based on GO functional annotation, the expressed sequences like Oxygen-evolving enhancer protein1, AARF domain-containing kinase protein, V-type proton ATPase subunitd2, RMD5 homologA, and ABC transporter G35 that are involved in photosynthesis, cellular transport, cell rescue and defense, polyubiquitination and secondary metabolism played a significant role implying a complex response to salt in S. maritima. This is the first report that SSH could facilitate screening across species and family specific identification of salt responsive genes provides insight into biological mechanisms underlying salinity response.

Probing the Energetic Metabolism of Resting Cysts under Different Conditions from Molecular and Physiological Perspectives in the Harmful Algal Blooms-Forming Dinoflagellate Scrippsiella trochoidea.

Energetic metabolism is essential in maintaining the viability of all organisms. Resting cysts play important roles in the ecology of dinoflagellates, particularly for harmful algal blooms (HABs)-causative species. However, the energetic metabolism underlying the germination potency maintenance of resting cysts of dinoflagellate have been extremely scarce in studies from physiological and, particularly, molecular perspectives. Therefore, we used the cosmopolitan Scrippsiella trochoidea as a representative of HABs-forming and cyst-producing dinoflagellates in this work to obtain novel insights into the molecular mechanisms, regulating the energetic metabolism in dinoflagellate resting cysts, under different physical condition. As the starting step, we established a cDNA subtractive library via suppression subtractive hybridization (SSH) technology, from which we screened an incomplete sequence for the β subunit of ATP synthase gene (β-F1-ATPase), a key indicator for the status of cell’s energetic metabolism. The full-length cDNA of β-F1-ATPase gene from S.trochoidea (Stβ-F1-ATPase) was then obtained via rapid amplification of cDNA ends (RACE) (Accession: MZ343333). Our real-time qPCR detections, in vegetative cells and resting cysts treated with different physical conditions, revealed that (1) the expression of Stβ-F1-ATPase in resting cysts was generally much lower than that in vegetative cells, and (2) the Stβ-F1-ATPase expressions in the resting cysts under darkness, lowered temperature, and anoxia, and during an extended duration of dormancy, were significantly lower than that in cysts under the condition normally used for culture-maintaining (a 12 h light:12 h dark cycle, 21 °C, aerobic, and newly harvested). Our detections of the viability (via Neutral Red staining) and cellular ATP content of resting cysts, at the conditions corresponding to the abovementioned treatments, showed that both the viability and ATP content decreased rapidly within 12 h and then maintained at low levels within the 4-day experimentation under all the three conditions applied (4 °C, darkness, and anoxia), which are well in accordance with the measurements of the transcription of Stβ-F1-ATPase. These results demonstrated that the energy consumption of resting cysts reaches a low, but somehow stable, level within a short time period and is lower at low temperature, darkness, and anoxia than that at ambient temperature. Our work provides an important basis for explaining that resting cysts survive long-term darkness and low temperature in marine sediments from molecular and physiological levels.

A novel lincRNA identified in buffalo oocytes with protein binding characteristics could hold the key for oocyte competence.

Studying the maternal oocyte-specific genes, in farm animals is a significant step towards delineating the underlying mechanisms that regulate oocyte quality, early embryonic development and survival. With the creation of buffalo oocyte-specific subtracted cDNA library, it has raised new questions which need to be answered. The present study has characterized one of the ESTs selected from the library and highlighted its importance in the oocyte quality. The selected EST was made full length by RLM-RACE and four transcript variants were identified. Bioinformatics analysis indicated the novelty of full-length transcript along with conserved intergenic nature. The largest transcript was identified as long intergenic noncoding RNA based upon coding potential calculator output. The expression analysis at different hours of oocyte maturation showed a significant variation in developmentally competent oocytes to that of incompetent ones. Along with this, the transcript was also found to have protein binding ability which was confirmed by RNA electrophoretic mobility shift assay. The protein used in the experiment was isolated from oocyte and cumulus cells via sonication. A novel lincRNA has been reported here that might have an important role in maturation of oocytes, inferred from its relative gene expression study and protein binding characteristics.

Ecotoxicogenomic analysis of zebrafish embryos exposed to triclosan and mixture triclosan and methyl triclosan using suppression subtractive hybridization and next-generation sequencing

Triclosan (TCS) and methyl-triclosan (MTCS), an environmental transformation product of biocide of TCS, have been detected in water, sediment, fish, and invertebrates. In this study, the key pathway perturbation in zebrafish (Danio rerio) embryos exposed to TCS (300 μg/L) and TCS/MTCS mixture (300 μg/L TCS + 30 μg/L MTCS) was assessed by integrating the metabolomic and transcriptomic dysregulation. The differential expressed genes (DEGs) were obtained from the subtracted cDNA libraries by using the suppression subtractive hybridization and next-generation sequencing approach. The dysregulation of twenty-eight GO terms and four KEGG pathways, including oxidative phosphorylation and cardiac muscle contraction, were shown in the TCS treatment group, indicating that TCS could disrupt the mitochondrial inner membrane function by downshifting the electrochemical gradient. Meanwhile, the addition of MTCS in the exposure would cause fourteen additional significant KEGG pathway changes, demonstrating the different effects between two exposure. A pathway-based analysis using the identified DEGs and the altered metabolites in zebrafish embryos treated with TCS and TCS/MTCS mixture, collectively, has been applied. This study demonstrated that the integration of SSH-NGS and metabolomics could reveal toxic effects and potential diseases associated with the exposures of TCS and MTCS in aquatic environments.

Overexpression of polygalacturonase-inhibiting protein (PGIP) gene from Hypericum perforatum alters expression of multiple defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens in tobacco

Hypericum perforatum L is a remarkable source of high-value secondary metabolites with increasing applications in pharmaceutical industry. However, improvement in the production of secondary metabolites through genetic engineering is a demanding task, as H. perforatum is not amenable to Agrobacterium tumefaciens-mediated transformation. In this study, we identified a Polygalacturonase-inhibiting protein (PGIP) gene from a subtractive cDNA library of A. tumefaciens-treated H. perforatum suspension cells. The role of HpPGIP in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum overexpressing HpPGIP alone or fused at the N-terminus to Phenolic oxidative coupling protein (Hyp-1), a gene that positively modulates resistance to A. tumefaciens. Furthermore, virus-induced gene silencing was employed to knock down the expression of the PGIP homologous in N. benthamiana. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in both HpPGIP and Hyp-1-PGIP transgenic plants, as assessed by GUS staining assays. However, silencing of PGIP in N. benthamiana increased the resistance to A. tumefaciens rather than susceptibility, which correlated with induction of pathogenesis-related proteins (PRs). The expression of core genes involved in several defense pathways was also analyzed in transgenic tobacco plants. Overexpression of HpPGIP led to up-regulation of key genes involved in hormone signaling, microRNA-based gene silencing, homeostasis of reactive oxygen species, and the phenylpropanoid pathway. Overexpression of Hyp-1-PGIP seemed to enhance the effect of PGIP on the expression of most genes analyzed. Moreover, HpPGIP was detected in the cytoplasm, nucleus and the plasma membrane or cell wall by confocal microscopy. Overall, our findings suggest HpPGIP modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

Novel ASR isolated from drought stress responsive SSH library in pearl millet confers multiple abiotic stress tolerance in PgASR3 transgenic Arabidopsis

A genomic resource of drought stress responsive genes/ESTs was generated using Suppression Subtractive Hybridization (SSH) approach in a drought stress tolerant Pennisetum glaucum genotype 841B. Fifty five days old plants were subjected to drought stress after withholding water for different time intervals (10 days, 15 days, 20 days and 25 days). A forward subtractive cDNA library was prepared from isolated RNA of leaf tissue. Differential gene expression under drought stress was validated for selected nine contigs by RT-qPCR. A transcript homologous to Setaria italica ASR3 upregulated under drought stress was isolated from genotype 841B and characterized. Heterologous expression of PgASR3 was validated in Arabidopsis and confirmed under multiple abiotic stress conditions. A total of four independent transgenic lines overexpressing gene PgASR3 were analyzed by Southern blot at T1 stage. For drought stress tolerance, three independent lines (T2 stage) were analyzed by biochemical and physiological assays at seedling stage. The growth rate (shoot and root length) of transgenic seedlings improved as compared to WT seedling under differenct abiotic stress conditions. The three transgenic lines were also validated for drought stress tolerance and RT-qPCR analysis, at maturity stage. Under drought stress conditions, the mature transgenic lines showed higher levels of RWC, chlorophyll and proline but lower levels of MDA as compared to WT plants. PgASR3 gene isolated and validated in this study can be utilized for developing abiotic stress tolerant crops.

Identification of salt-responsive genes from C4 halophyte Suaeda nudiflora through suppression subtractive hybridization and expression analysis under individual and combined treatment of salt and elevated carbon dioxide conditions

Salinization of soil is a prime abiotic stress that limits agriculture productivity worldwide. To Study the mechanisms that halophytes take up to survive under high salt condition is important in engineering salinity stress tolerance in sensitive species. Suaeda nudiflora is a halophyte plant that grows in the saline environment and extreme high tidal belt. The species have high capability to produce high protein biomass in salty soils due to C4 photosynthesis. The physiological and biochemical changes in S. nudiflora under salinity stress were studied by measuring chlorophyll content, electrolytic leakage, level of lipid peroxidation and total soluble sugars. Increased lipid peroxidation and electrolytic leakage was observed in salt stressed S. nudiflora compared to control plants. A suppression subtractive hybridization strategy was employed to identify differentially expressed genes under salt treatment in S. nudiflora. A total of 333 positive clones were identified and screened. Of these, 250 expressed sequence tags were identified. cDNA subtraction library resulted in 33 contigs and 138 singletons. The functional annotation and metabolic pathways identification were performed using the Blast2GO program. In addition, we analyzed the expression patterns of 18 genes associated with salt stress-responsive pathways by semi-quantitative PCR under salt and elevated carbon dioxide (CO2) conditions. Several of the analyzed genes showed an increase in expression levels under different time points of salt treatment and at different concentrations of salt. When the same genes were studied for its expression under elevated CO2 concentrations, most of the known salt responsive genes showed higher expression under the combined treatment of elevated CO2 concentrations (500ppm) and NaCl treatment (200mM) compare to ambient condition. This implies that salt responsive genes are enhanced at elevated CO2 concentrations.

Transgenic expression of Hyp-1 gene from Hypericum perforatum L. alters expression of defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens.

Phenolic oxidative coupling protein (Hyp-1) isolated from Hypericum perforatum L. was characterized as a defense gene involved in H. perforatum recalcitrance to Agrobacterium tumefaciens-mediated transformation Hypericum perforatum L. is a reservoir of high-value secondary metabolites of increasing interest to researchers and to the pharmaceutical industry. However, improving their production via genetic manipulation is a challenging task, as H. perforatum is recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, phenolic oxidative coupling protein (Hyp-1), a pathogenesis-related (PR) class 10 family gene, was selected from a subtractive cDNA library from A. tumefaciens-treated H. perforatum suspension cells. The role of Hyp-1 in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum and Lactuca sativa overexpressing Hyp-1, and in Catharanthus roseus silenced for its homologous Hyp-1 gene, CrIPR. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in Hyp-1 transgenic plants. However, silencing of CrIPR induced CrPR-5 expression and decreased expression efficiency of Agrobacterium. The expression of core genes involved in several defense pathways was also analyzed in Hyp-1 transgenic tobacco plants. Overexpression of Hyp-1 led to an ample down-regulation of key genes involved in auxin signaling, microRNA-based gene silencing, detoxification of reactive oxygen species, phenylpropanoid pathway and PRs. Moreover, Hyp-1 was detected in the nucleus, plasma membrane and the cytoplasm of epidermal cells by confocal microscopy. Overall, our findings suggest Hyp-1 modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

Ectopic Expression of Cold Responsive LlaCIPK Gene Enhances Cold Stress Tolerance in Nicotiana tabacum.

Low-temperature stress severely affects the growth, development, and geographical distribution of various crop plants, resulting in significant economic loss to producers. In a quest to identify cold-regulated genes, we constructed a cDNA suppression subtractive library from a high altitude adapted ecotype of Lepidium. We cloned a cold-induced gene LlaCIPK from the subtracted cDNA library which gave homology to Arabidopsis CIPK15 gene. The predicted 3D structure of LlaCIPK protein also showed homology with Arabidopsis CIPK protein. Quantitative real-time PCR analysis in Lepidium seedlings exposed to 6 h of cold stress shows a 3-fold increase in the expression of LlaCIPK transcript. The expression of LlaCIPK was also differentially regulated by ethylene, CaCl2, ABA, and SA treatments. Ethylene and CaCl2 treatments up regulated LlaCIPK expression, whereas ABA and SA treatments down regulated the LlaCIPK expression. Transgenic plants overexpressing LlaCIPK gene under constitutive promoter show an increased level of proline and cell membrane stability. Taken together, our results suggest that the LlaCIPK contributes to the cold-response pathway in Lepidium plants.

Identification of the LEA family members from Caragana korshinskii (Fabaceae) and functional characterization of CkLEA2-3 in response to abiotic stress in Arabidopsis

Plants are exposed to different abiotic stresses that affect growth, development, and productivity. Previous studies have shown that late embryogenesis abundant (LEA) proteins play important roles in plant adaptation to abiotic stresses. However, reports that demonstrate their biological functions, especially in Caragana korshinskii Kom., are still very limited. In this study, 26 LEA genes were identified from dehydration-treated suppressive subtractive hybridizations cDNA library and transcriptome sequencing data of C. korshinskii, and were classified into seven groups according to their structural features. Quantitative real-time PCR analysis revealed that the CkLEAs were induced by abscisic acid (ABA) and diverse abiotic stresses and widely expressed in various tissues. Moreover, overexpression of CkLEA2-3 in Arabidopsis thaliana L. Heynh resulted in enhanced tolerance to ABA treatment, and osmotic and salt stresses during seed germination. CkLEA2-3 overexpression lines also exhibited resistance to drought stress during seedling development. Taken together, our results indicate that CkLEA2-3 plays positive roles in conferring abiotic stress tolerance in Arabidopsis.

Molecular cloning and characterization of the β-1,3-glucan recognition protein in Anatolica polita

β-1,3-Glucan recognition protein (βGRP) is an important pattern recognition protein, which could trigger immune response to eliminate pathogens by identifying and combining the pathogenic bacteria. In the present study, a β-1,3-glucan recognition protein gene (ApβGRP) was cloned from a desert beetle Anatolica polita based on the EST sequence of ApβGRP in the suppression subtractive cDNA library. Quantitative real-time PCR (qRT-PCR) results showed that ApβGRP transcript in A. polita was significantly upregulated under the challenge of Escherichia coli and Staphylococcus aureus. Western blot analysis indicated that recombinant ApβGRP expressed in E. coli BL21, has the ability of binding to E. coli and S. aureus. Moreover, agglutination assay suggested that recombinant ApβGRP could agglutinate E. coli, S. aureus and Saccharomyces cerevisiae. The predicted 3D structure showed that ApβGRP possesses a typical β-glucan recognition domain with seven β-strands structures and conserved amino acid sequence. These data indicate that ApβGRP may be involved in immune defense in A. polita and could recognize and bind the bacteria against the invasion of external pathogens.

Further confirmation of second- and third-generation Eimeria necatrix merozoite DEGs using suppression subtractive hybridization.

In our previous study, we obtained a large number of differentially expressed genes (DEGs) between second-generation merozoites (MZ-2) and third-generation merozoites (MZ-3) of Eimeria necatrix using RNA sequencing (RNA-seq). Here, we report two subtractive cDNA libraries for MZ2 (forward library) and MZ3 (reverse library) that were constructed using suppression subtractive hybridization (SSH). PCR amplification revealed that the MZ2 and MZ3 libraries contained approximately 96.7% and 95% recombinant clones, respectively, and the length of the inserted fragments ranged from 0.5 to 1.5kb. A total of 106 and 111 unique sequences were obtained from the MZ2 and MZ3 libraries, respectively, and were assembled into 13 specific consensus sequences (contigs or genes) (5 from MZ2 and 8 from MZ3). The qRT-PCR results revealed that 11 out of 13 genes were differentially expressed between MZ-2 and MZ-3. Of 13 genes, 11 genes were found in both SSH and our RNA-seq data and displayed a similar expression trend between SSH and RNA-seq data, and the remaining 2 genes have not been reported in both E. necatrix genome and our RNA-seq data. Among the 11 genes, the expression trends of 8 genes were highly consistent between SSH and our RNA-seq data. These DEGs may provide specialized functions related to the life-cycle transitions of Eimeria species.

Genomic technologies for Hevea breeding.

The commercial production of high quality natural rubber (NR) solely depends on Hevea brasiliensis Muell. Arg, (Para rubber tree) and accounts for >98% of total production worldwide. NR with its unique properties is an essential commodity for the automobile industry and its synthetic counterparts are in no way substitute to it. The rubber tree genome is very complex and plays an important role in delivering the unique properties of Hevea. But a lack of knowledge on the molecular mechanisms of rubber biosynthesis, disease resistance, etc., in elite clones of rubber still persists. Marker-assisted selection and transgenic techniques were proved to be advantageous in improving the breeding efficiency for latex yield, disease resistance, etc. The suppression subtractive hybridization (SSH), in the form of subtracted cDNA libraries and microarrays, can assist in searching the functions of expressed genes (candidate gene approach). Expressed sequence tags (ESTs) related to various metabolic aspects are well utilized to create EST banks that broadly represent the genes expressed in one tissue, such as latex cells, that assists in the study of gene function and regulation. Transcriptome analysis and gene mapping have been accomplished in Hevea at various stages. However, a selection criterion to delineate high yielding genotypes at the juvenile stage has not been accomplished so far. This is the main pit fall for rubber breeding apart from stock-scion interactions leading to yield differences among a clonally multiplied population. At least four draft genome sequences have been published on Hevea rubber, and all give different genome size and contig lengths-a comprehensive and acceptable genomic map remains unfulfilled. The progress made in molecular markers, latex biosynthesis genes, transcriptome analysis, chloroplast and mitochondrial DNA diversity, paternity identification through Breeding without Breeding (BwB), stimulated latex production and its molecular intricacies, molecular biology of tapping panel dryness, genomics for changed climates and genome mapping are discussed in this review. These information can be utilized to improvise the molecular breeding programs of Hevea in future.

Heat stress transcripts, differential expression, and profiling of heat stress tolerant gene TaHsp90 in Indian wheat (Triticum aestivum L.) cv C306.

To generate a genetic resource of heat stress responsive genes/ESTs, suppression subtractive hybridization (SSH) library was constructed in a heat and drought stress tolerant Indian bread wheat cultivar C306. Ninety three days old plants during grain filling stage were subjected to heat stress at an elevated temperature of 37°C and 42°C for different time intervals (30 min, 1h, 2h, 4h, and 6h). Two subtractive cDNA libraries were prepared with RNA isolated from leaf samples at 37°C and 42°C heat stress. The ESTs obtained were reconfirmed by reverse northern dot blot hybridization. A total of 175 contigs and 403 singlets were obtained from 1728 ESTs by gene ontology analysis. Differential expression under heat stress was validated for a few selected genes (10) by qRT-PCR. A transcript showing homology to Hsp90 was observed to be upregulated (7.6 fold) under heat stress in cv. C306. CDS of TaHsp90 (Accession no. MF383197) was isolated from cv. C306 and characterized. Heterologous expression of TaHsp90 was validated in E. coli BL21 and confirmed by protein gel blot and MALDI-TOF analysis. Computational based analysis was carried out to understand the molecular functioning of TaHsp90. The heat stress responsive SSH library developed led to identification of a number of heat responsive genes/ESTs, which can be utilized for unravelling the heat tolerance mechanism in wheat. Gene TaHsp90 isolated and characterized in the present study can be utilized for developing heat tolerant transgenic crops.

Prognostic and predictive value of long non-coding RNA GAS5 and mircoRNA-221 in colorectal cancer and their effects on colorectal cancer cell proliferation, migration and invasion.

LncRNA and microRNA play an important role in the development of human cancers; they can act as a tumor suppressor gene or an oncogene. LncRNA GAS5, originating from the separation from tumor suppressor gene cDNA subtractive library, is considered as an oncogene in several kinds of cancers. The expression of miR-221 affects tumorigenesis, invasion and metastasis in multiple types of human cancers. However, there's very little information on the role LncRNA GAS5 and miR-221 play in CRC. Therefore, we conducted this study in order to analyze the association of GAS5 and miR-221 with the prognosis of CRC and preliminary study was done on proliferation, metastasis and invasion of CRC cells. In the present study, we demonstrate the predictive value of long non-coding RNA GAS5 (lncRNA GAS5) and mircoRNA-221 (miR-221) in the prognosis of colorectal cancer (CRC) and their effects on CRC cell proliferation, migration and invasion. One hundred and fifty-eight cases with CRC patients and 173 cases of healthy subjects that with no abnormalities, who've been diagnosed through colonoscopy between January 2012 and January 2014 were selected for the study. After the clinicopathological data of the subjects, tissue, plasma and exosomes were collected, lncRNA GAS5 and miR-221 expressions in tissues, plasma and exosomes were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The diagnostic values of lncRNA GAS5 and miR-221 expression in tissues, plasma and exosomes in patients with CRC were analyzed using receiver operating characteristic curve (ROC). Lentiviral vector was constructed for the overexpression of lncRNA GAS5, and SW480 cell line was used for the transfection of the experiment and assigned into an empty vector and GAS5 groups. The cell proliferation, migration and invasion were tested using a cell counting kit-8 assay and Transwell assay respectively. The results revealed that LncRNA GAS5 was upregulated while the miR-221 was downregulated in the tissues, plasma and exosomes of patients with CRC. The results of ROC showed that the expressions of both lncRNA GAS5 and miR-221 in the tissues, plasma and exosomes had diagnostic value in CRC. While the LncRNA GAS5 expression in tissues, plasma and exosomes were associated with the tumor node metastasis (TNM) stage, Dukes stage, lymph node metastasis (LNM), local recurrence rate and distant metastasis rate, the MiR-221 expression in tissues, plasma and exosomes were associated with tumor size, TNM stage, Dukes stage, LNM, local recurrence rate and distant metastasis rate. LncRNA GAS5 and miR-221 expression in tissues, plasma and exosomes were found to be independent prognostic factors for CRC. Following the overexpression of GAS5, the GAS5 expressions was up-regulated and miR-221 expression was down-regulated; the rate of cell proliferation, migration and invasion were decreased.

Integrated miRNA-mRNA analysis reveals regulatory pathways underlying the curly fleece trait in Chinese tan sheep

BackgroundTan sheep is an indigenous Chinese breed well known for its beautiful curly fleece. One prominent breed characteristic of this sheep breed is that the degree of curliness differs markedly between lambs and adults, but the molecular mechanisms regulating the shift are still not well understood. In this study, we identified 49 differentially expressed (DE) microRNAs (miRNAs) between Tan sheep at the two stages through miRNA-seq, and combined the data with that in our earlier Suppression Subtractive Hybridization cDNA (SSH) library study to elucidate the mechanisms underlying curly fleece formation.ResultsThirty-six potential miRNA-mRNA target pairs were identified using computational methods, including 25 DE miRNAs and 10 DE genes involved in the MAPK signaling pathway, steroid biosynthesis and metabolic pathways. With the differential expressions between lambs and adults confirmed by qRT-PCR, some miRNAs were already annotated in the genome, but some were novel miRNAs. Inhibition of KRT83 expression by miR-432 was confirmed by both gene knockdown with siRNA and overexpression, which was consistent with the miRNAs and targets prediction results.ConclusionOur study represents the comprehensive analysis of mRNA and miRNA in Tan sheep and offers detailed insight into the development of curly fleece as well as the potential mechanisms controlling curly hair formation in humans.

Identification of Anther-Specific Genes from Sesame and Functional Assessment of the Upstream Region of a Tapetum-Specific β-1,3-glucanase Gene

Sesamum indicum is widely cultivated in India for its superior agronomic characters like adaptation to the warm climate of India and resistance to drought and pathogen attack. However, the yield of sesame per unit area of land is quite low. In this context, the genes involved with reproductive tissue development is very important since they are directly involved with crop productivity. Therefore, identification and isolation of anther-specific genes through PCR-Select cDNA subtraction between cDNAs of anthers and leaves was carried out. Sequencing of ~ 700 clones from the cDNA subtraction library followed by BLASTX analysis using sesame database resulted in the identification of 163 annotated unique transcripts, of which 18 transcripts showed homology with the genes of exclusive anther-/pollen-specific functions. Furthermore, anther-specific expression of some of the sesame transcripts as well as already identified anther-specific genes of Arabidopsis and rice have been monitored during development of sesame anthers. Subsequently, full-length cDNA sequence and the upstream sequence of an anther-specific β-1 3-glucanase gene was isolated. Transgenic tobacco plants were developed with GUS gene under the control of a 437-bp upstream sequence along with the first exon and first intron of the gene. Monitoring of GUS gene expression pattern indicated exclusive expression of the promoter in the tapetal cell layer before tetrad formation in the anther. Interestingly, upon treatment with ABA as well as drought stress, GUS was found to express in the vegetative parts of the plant such as leaves and roots as well.

Gene expression profiling of upregulated mRNAs in granulosa cells of bovine ovulatory follicles following stimulation with hCG

BackgroundOvulation and luteinization of follicles are complex biological processes initiated by the preovulatory luteinizing hormone surge. The objective of this study was to identify genes that are differentially expressed in bovine granulosa cells (GC) of ovulatory follicles.MethodsGranulosa cells were collected during the first follicular wave of the bovine estrous cycle from dominant follicles (DF) and from ovulatory follicles (OF) obtained 24 h following injection of human chorionic gonadotropin (hCG). A granulosa cell subtracted cDNA library (OF-DF) was generated using suppression subtractive hybridization and screened.ResultsDetection of genes known to be upregulated in bovine GC during ovulation, such as ADAMTS1, CAV1, EGR1, MMP1, PLAT, PLA2G4A, PTGES, PTGS2, RGS2, TIMP1, TNFAIP6 and VNN2 validated the physiological model and analytical techniques used. For a subset of genes that were identified for the first time, gene expression profiles were further compared by semiquantitative RT-PCR in follicles obtained at different developmental stages. Results confirmed an induction or upregulation of the respective mRNAs in GC of OF 24 h after hCG-injection compared with those of DF for the following genes: ADAMTS9, ARAF, CAPN2, CRISPLD2, FKBP5, GFPT2, KIT, KITLG, L3MBLT3, MRO, NUDT10, NUDT11, P4HA3, POSTN, PSAP, RBP1, SAT1, SDC4, TIMP2, TNC and USP53. In bovine GC, CRISPLD2 and POSTN mRNA were found as full-length transcript whereas L3MBLT3 mRNA was alternatively spliced resulting in a truncated protein missing the carboxy-terminal end amino acids, 774KNSHNEL780. Conversely, L3MBLT3 is expressed as a full-length mRNA in a bovine endometrial cell line. The 774KNSHNEL780 sequence is well conserved in all mammalian species and follows a SAM domain known to confer protein/protein interactions, which suggest a key function for these amino acids in the epigenetic control of gene expression.ConclusionsWe conclude that we have identified novel genes that are upregulated by hCG in bovine GC of OF, thereby providing novel insight into peri-ovulatory regulation of genes that contribute to ovulation and/or luteinization processes.